Preservation and storage medium for biological materials

ABSTRACT

A protectant mixture for use in preserving biological materials comprising (1) at least one polyhydroxy compound, where the total amount of polyhydroxy compound in the mixture is from about 5% to about 60% by weight of the mixture where the mixture is an aqueous solution and is from about 10% to about 95% where the mixture is in solid form, and (2) phosphate ions, where the total amount of phosphate ions in the mixture is such that the molar ratio of phosphate ions to hydroxy groups in the polyhydroxy compound is from about 0.025 to about 0.625; a preservation medium comprising (1) a biological material, (2) at least one polyhydroxy compound, where the total amount of polyhydroxy compound in the medium is from about 5% to about 60% by weight of the medium, and (3) phosphate ions, where the total amount of phosphate ions in the mixture is such that the molar ratio of phosphate ions to hydroxy groups in the polyhydroxy compound is from about 0.025 to about 0.625; methods of preserving the preservation medium; and the resulting preserved biological material composition.

FIELD AND BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to preserving andstabilizing biological materials by freezing, drying, and freeze-drying,and more specifically to protectant mixtures and aqueous preservationmedia for preserving biological materials, as well as methods ofpreserving biological materials and the preserved biological materialcompositions themselves.

[0002] The preservation of the structure and function of biologicalmolecules is of fundamental importance to biology, biochemistry, andmedicine. Biological materials, such as proteins, enzymes, cells,tissues, nucleic acid, semen, blood and its components, mammalianorgans, and foodstuffs must often be stored and preserved for later use.Preservation of these biological materials is usually achieved by eitherfreezing or drying, or a combination of the two processes. There areseveral commonly-used drying techniques: drying by evaporation into amoving gas stream (ambient air-drying), drying under vacuum at ambienttemperatures (vacuum-drying), or drying by contacting a fine mist ofdroplets with warm air (spray-drying). Simple freezing is often donewhen drying is either harmful or unnecessary. Certain biologicalmaterials are best preserved by freeze-drying (lyophilization), atwo-step process in which the sample is first frozen and then dried atlow temperature under vacuum.

[0003] The structure and function of most biological materials isdependent upon their aqueous environment. Therefore, changes to theiraqueous environment resulting from freezing and drying processes canoften have drastic consequences for a biological material. Furthermore,freeze-drying combines the stresses due to both freezing and drying. Thefreezing step of this process can have undesirable side effects, such asthe denaturation of proteins and enzymes, and rupture of cells. Theseeffects result from mechanical, chemical, and osmotic stresses inducedby crystallization of ice in these materials. As a result, the activityof the biological material upon rehydration is lost either in itsentirety, or to such a significant extent that the material is no longeruseful for its intended purpose.

[0004] To prevent or reduce the adverse effects upon reconstitution orrehydration, protective agents, such as cryoprotectants orlyoprotectants (freeze-drying) are used. For such protective agents tobe effective, they must be non-toxic to the biological material at theconcentrations encountered during the preservation process, and mustinteract favorably with water and with the biological material. Variousprotective agents have been used in the art, with varying degrees ofsuccess. These include fish proteins, certain polymers, skim milk,glycerol, dimethyl sulfoxide, and disaccharides, such as trehalose.Unfortunately, suitable protective agents and cryopreservation protocolshave been developed only for a limited number of systems.

[0005] Disaccharides, such as sucrose and trehalose, are naturalcryoprotectants. Trehalose is a particularly attractive cryoprotectantbecause it has actually been isolated from plants and animals thatremain in a state of suspended animation during periods of drought.Trehalose has been shown to be an effective protectant for a variety ofbiological materials, both in ambient air-drying and freeze-drying.Research has shown, (see Crowe, J. H., Crowe., L. M., and Mouriadian,R., Cryobiology, 20, 346-356 (1983)), that liposomes dried in thepresence of trehalose retain both their functional and structuralintegrity upon rehydration. U.S. Pat. No. 5,556,771 discloses the use oftrehalose, or trehalose in combination with polyvinylpyrrolidone topreserve reverse transcriptase and RNA polymerase. U.S. Pat. No.5,512,547 discloses the use of trehalose to preserve botulinumneurotoxin. Likewise, U.S. Pat. No. 4,891,319 discloses a method ofprotecting proteins and other biological macromolecules, such asenzymes, serum, serum complement, antibodies, antigens, fluorescentproteins and vaccine components using trehalose. Specifically, anaqueous mixture containing the macromolecule and trehalose is dried at atemperature above freezing in the presence of 0.05 to about 20%trehalose by weight of the aqueous system.

[0006] However, there are some drawbacks associated with the use oftrehalose as the sole cryoprotectant. To preserve many biologicalmaterials by freeze-drying, large amounts of trehalose must be used;concentrations of trehalose greater than 60% by weight of a givenpreservation medium are sometimes necessary. This is costly. Further, ahigh concentration of trehalose reduces the solubility of other solutesin the system.

[0007] Thus, it has been proposed to use trehalose in combination with apolymeric gelling agent, such as carboxymethylcellulose orcarboxyethylcellulose. It has been suggested for human blood thatsaccharides combined with polymers are even more effectivecryoprotectants than pure trehalose. See U.S. Pat. No. 5,171,661;Sutton, R. L., J. Chem. Soc. Faraday Trans., 87, 3747 (1991).Unfortunately, attempts to confirm the beneficial effect of the gellingagents have been unsuccessful. (G. Spieles, I.

[0008] Heschel, and G. Rau, Cryo-Letters 17, 43-52 (1996), J. H. Crowe,A. E. Oliver, F. A. Hoekstra, and L. M. Crowe, Cryobiology 35, 20-30(1997).). Moreover, this protective combination cannot be used formedical purposes, because the polymer gelling agents are not acceptedwell by the human body. As a result, this combination is not veryuseful, and does not provide much, if any, practical improvement overthe use of trehalose alone.

[0009] Another, more serious problem associated with the use oftrehalose is that biological materials preserved using trehalose aloneare not storage stable for extended periods of time, especially thosestored at superambient temperatures and/or in humid environments. Inother words, biological materials preserved with trehalose can losetheir activity in a matter of hours or days, depending on the humidityand temperature of the storage conditions.

[0010] Therefore, at present, freeze-drying with trehalose is of limiteduse for extended term storage of biological materials, such as proteins,enzymes, cells, tissues, nucleic acid, semen, blood and its components,mammalian organs, and foodstuffs, over a wide range of storageconditions, because the material will degrade, and will not havesufficient activity upon reconstitution. From a practical standpoint,this is clearly unacceptable for medical products, as one of the reasonsfor preserving the materials in the first place is to provide astorage-stable product.

[0011] Nor can many of the various room temperature drying techniques beeffectively used at present. These methods, while less complicated andless costly than freeze-drying, are generally more destructive tobiological materials. Many biological materials are more prone to grossconformational changes and unwanted reactions when preserved usingmethods that take place at ambient temperature than when freeze-dryingis used. As a result, even where presently known protective agents areused, the activity of many rehydrated biological materials is bothunsatisfactory in its own right, and significantly less than ifpreserved by freeze-drying.

[0012] Thus, a need exists for a protectant mixture that is useful for awide range biological materials. A further need exists for a protectantmixture that can be effectively used in both freeze-drying processes anddrying processes involving ambient-temperature drying. There is also aneed for a protectant mixture that is less costly than those presentlybeing used. Finally, and very importantly, there is a need for aprotectant mixture that provides stable media for preservation ofbiological materials over extended periods of time at elevatedtemperatures and varying degrees of humidity, which can be encounteredduring shipping and storage of materials, while still retaining asignificant amount of activity upon rehydration.

[0013] All of these needs are met by the protectant mixture, aqueousprotective medium and resulting preserved biological materialcompositions of the present invention.

SUMMARY OF THE INVENTION

[0014] It has been found that a protectant mixture for use in preservingbiological materials, comprising: (a) at least one polyhydroxy compound,where the total amount of polyhydroxy compound in the mixture is fromabout 5% to about 60% by weight of the mixture if the mixture is anaqueous solution and from about 10% to about 95% by weight if themixture is in solid form; and (b) phosphate ions, where the total amountof phosphate ions in the medium is such that the molar ratio ofphosphate ions to hydroxyl groups in the polyhydroxy compound is fromabout 0.025 to about 0.625, can be used with a wide variety ofbiological materials to provide an aqueous preservation medium. Thisaqueous preservation medium can then be used in a multiplicity ofpreservation processes, including freezing, freeze-drying and otherdrying processes, such as spray-drying, vacuum-drying, or ambientair-drying, to provide a stable, preserved composition of the biologicalmaterial of interest. This preserved composition is stable for extendedperiods of time at superambient temperatures and/or relative humidity.Further, when the preserved biological material composition isrehydrated, the structural and functional integrity of the preservedbiological material has been retained to such an extent that thebiological material can be used for its intended purpose.

[0015] Therefore, the present invention also provides a method forpreparing a preserved biological material composition from theabove-noted preservation medium, as well as the preserved biologicalmaterial composition itself.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a graph of percent activity recovered from freeze-driedlactate dehydrogenase compositions made using varying amounts and molarratios of phosphate and trehalose.

[0017]FIG. 2 is a graph of percent activity recovered from frozenlactate dehydrogenase compositions made using varying amounts and molarratios of phosphate and trehalose.

[0018]FIG. 3 is a graph of percent activity recovered over time fromfreeze-dried lactate dehydrogenase compositions made using varyingamounts and molar ratios of phosphate and trehalose.

[0019]FIG. 4 is a graph of percent activity recovered over time fromfreeze-dried lactate dehydrogenase compositions made using varyingamounts and molar ratios of phosphate and trehalose.

[0020]FIG. 5 is a graph of percent activity recovered over time fromfreeze-dried lactate dehydrogenase compositions made using varyingamounts and molar ratios of phosphate and trehalose.

[0021]FIG. 6 is a graph of glass transition temperatures for variousprotectant mixtures of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention is based on the remarkable discovery thatbiological materials can be preserved while retaining substantialactivity, when the biological material is combined with the protectantmixture of the present invention to form an aqueous preservation medium,which in turn is formed into a preserved biological material compositionby subjecting the aqueous preservation medium of the present inventionto (1) various drying techniques, including freeze-drying, ambientair-drying, vacuum-drying, and spray-drying, or (2) other preservationmethods known in the art, such as freezing. The protectant mixture ofthe present invention comprises: (a) at least one polyhydroxy compound,where the total amount of polyhydroxy compound in the mixture is fromabout 5% to about 60% by weight of the mixture if the mixture is anaqueous solution and from about 10% to about 95% by weight if themixture is in solid form; and (b) phosphate ions, where the total amountof phosphate ions in the mixture is such that the molar ratio ofphosphate ions to hydroxyl groups in the polyhydroxy compound is fromabout 0.025 to about 0.625. The aqueous preservation medium of thepresent invention comprises: (a) a biological material; (b) at least onepolyhydroxy compound, where the total amount of polyhydroxy compound inthe medium is from about 5% to about 60% by weight of the medium; and(c) phosphate ions, where the total amount of phosphate ions in themedium is such that the molar ratio of phosphate ions to hydroxyl groupsin the polyhydroxy compound is from about 0.025 to about 0.625.

[0023] Biological Materials

[0024] A wide range of biological materials can be used with theinventive protectant mixtures to form the aqueous preservation medium ofthe present invention. This preservation medium can then be subjected tothe processes of the present invention to make a preserved biologicalmaterial composition. These biological materials, include, withoutlimitation:

[0025] (a) enzymes, such as lactate dehydrogenase andphosphofructokinase;

[0026] (b) proteins, such as insulin;

[0027] (c) serum complement;

[0028] (d) vaccines;

[0029] (e) tissue, including skin, veins and arteries;

[0030] (f) viruses, such as adenovirus;

[0031] (g) mammalian organs, such as the liver, pancreas and lungs;

[0032] (h) blood, and its components, including red blood cells, whiteblood cells and platelets;

[0033] (i) cells, including prokaryotic cells (including bacteria) andeukaroytic cells;

[0034] (j) semen;

[0035] (k) other biological materials, including nucleic acids, andlipid vesicles; and

[0036] (l) foodstuffs.

[0037] The above is merely exemplary of some of the myriad of biologicalmaterials that can be made into the preserved biological materialcompositions of the claimed invention using the protectant mixture,aqueous preservation medium and process of the claimed invention. Anybiological material for which preservation for later use is desirablecan be used with the protectant mixture, to form the preservation media,which can then be preserved by the preservation methods of the inventionto form preserved compositions.

[0038] Polyhydroxy Compound

[0039] Polyhydroxy compounds useful in the present invention includenatural and synthetic monosaccharides and polysaccharides, othercarbohydrates, and polyalcohols and their derivatives. Here,“polysaccharides” are defined as saccharides containing two or moremonosaccharide units. The individual polyhydroxy compounds can be usedsingly, or in combination with other types of polyhydroxy compounds.From the wide variety of useful polyhydroxy compounds, the use ofmonosaccharides and polysaccharides is preferred. Of the saccharides,disaccharides, such as trehalose, maltose, lactose, and sucrose arepreferred for use in the present invention, with trehalose being mostpreferred.

[0040] The amount of polyhydroxy compound present in the protectantmixture, preservation medium, and preserved composition of the presentinvention depends upon the specific polyhydroxy compounds, andbiological material selected for use, as well as the mass of biologicalmaterial being preserved. This can be adjusted and optimized for a givensystem.

[0041] Generally, the polyhydroxy compounds are present in theprotectant mixture of the present invention in a total amount of fromabout 5% to about 60% by weight of the mixture, where the mixture is anaqueous solution. Where the protectant mixture is supplied as a solid,for example as a powder, the polyhydroxy compounds should be present ina total amount of from about 10% to about 95% by weight of the mixture,with an amount in the range of about 20% to about 95% by weight of themixture being preferred. Where the protectant mixture is an aqueoussolution, the polyhydroxy compounds are preferably present in a totalamount such that the total amount of polyhydroxy compound in the mixtureis from about 5% to about 40% by weight of the mixture, with an amountin the range of about 10% to about 30% by weight of the mixture beingparticularly preferred.

[0042] Likewise, the polyhydroxy compounds should be present in thepreservation medium of the present invention in an amount such that thetotal amount of polyhydroxy compounds in the aqueous preservation mediumis from about 5% to about 60% by weight of the aqueous preservationmedium. Preferably, the total amount of polyhydroxy compound presentshould be from about 5% to about 40% by weight of the aqueouspreservation medium, with an amount in the range of about 10% to about30% by weight of the aqueous preservation medium being particularlypreferred.

[0043] It should be emphasized that the above ranges can be varied, forexample, depending upon the amount of biological material in thepreservation medium and the preservation method chosen for use.

[0044] Use of the above amounts of polyhydroxy compound in the aqueouspreservation medium of the present invention will upon partial orcomplete removal of liquid water, result in a preserved biologicalmaterial composition having from about 5% to about 95% polyhydroxycompound by weight of the composition. Again, this amount will depend onthe mass of the biological material being preserved, the amount ofphosphate present, and the amount of water removed from the systemduring preservation. The amount of polyhydroxy compound in the preservedbiological composition can be determined from the amount present in theprotectant mixture and/or aqueous preservation medium. Alternatively,the amount of polyhydroxy compound in the preserved biological materialcomposition can be determined by analytical methods known in the art,such as column chromatography.

[0045] Phosphate Ions

[0046] Any source of phosphate ions can be used in the protectantmixture, preservation medium, preservation process, and preservedcomposition of the present invention. While not wishing to be bound byany particular theory, it is believed that the phosphate ions form acomplex with the polyhydroxy compound, which may contain severalmolecules of the polyhydroxy compound in a three-dimensionalsupermolecular structure cross-linked by the phosphate ions. The aqueouspreservation medium has a much higher viscosity than a system containingthe polyhydroxy compound alone in the same amount, and the preservedbiological material composition has a higher glass transitiontemperature (T_(g)) than a composition containing only the polyhydroxycompound.

[0047] As stated earlier, the phosphate ions can be provided from anysource, including acids and salts. The use of sodium and potassium saltsare preferred. Potassium salts are most preferred, because they haveexcellent solubility characteristics at low temperature, and crystallizeas anhydrous crystals. Therefore, the phosphate ions are preferablyprovided through the use of sodium and/or potassium salts, with the useof a mixture of monobasic and dibasic potassium phosphate beingparticularly preferred.

[0048] The amount of phosphate ion that is optimal for a givenprotectant mixture and/or preservation medium depends on severalvariables, including the specific biological material to be preserved,the amount and type of polyhydroxy compound in the protectant mixtureand/or preservation medium, and the amount of biological material in thesystem. Generally, phosphate ions should be present in the protectantmixture and/or aqueous preservation medium in a total amount such thatthe molar ratio of phosphate ions to hydroxyl groups in the polyhydroxycompound is from about 0.025 to about 0.625. Preferably, the phosphateions are present in an amount such that the molar ratio of phosphateions to hydroxyl groups in the polyhydroxy compound is from about 0.0375to about 0.625.

[0049] The molar ratio of phosphate ions to hydroxyl groups in thepolyhydroxy compound will remain substantially constant throughout thepreservation process, resulting in a preserved biological materialhaving substantially the same molar ratio of phosphate ions to hydroxylgroups in the polyhydroxy compound. Thus, the amount of phosphate ionspresent in the preserved biological material composition followsdirectly from the amount present in the aqueous preservation medium.Alternatively, the amount of phosphate ions in the preserved biologicalmaterial composition can be determined analytically by methods known inthe art, including ion chromatography and chemiluminescence.

[0050] The useful amount of phosphate ions can also be determined on thebasis of moles of phosphate ion per moles of the polyhydroxy compound bymultiplying the above ratios by the number of hydroxyl groups presentper mole of the polyhydroxy compound being used. For example, trehaloseand sucrose have 8 moles of hydroxyl groups per mole of compound.Therefore, where trehalose or sucrose is used as the polyhydroxycompound, the ratio of from about 0.025 to about 0.625 moles ofphosphate ions per mole of hydroxyl groups can be achieved by addingsufficient phosphate ions to achieve a molar ratio of phosphate ions tosucrose or trehalose of about 0.2 to about 5. For trehalose or sucrose,the preferred molar ratio of from about 0.0375 to about 0.625 translatesto a molar ratio of phosphate ions to sucrose or trehalose of from about0.3 to about 5.

[0051] It has been found that the effectiveness of the phosphate ions instabilizing and preserving the structure and function of a givenbiological material increases as the molar ratio of phosphate ions tohydroxyl groups increases from zero, but only up to a point (the optimumratio), after which the use of additional phosphate ion provides no oronly a slight increase in effectiveness. As discussed previously, theoptimal ratio depends on several factors, including the amount and typeof biological material used, the amount and type of polyhydroxycompounds in the preservation medium, the pH of the preservation medium,and the preservation technique to be utilized.

[0052] The use of phosphate ions at a molar ratio of phosphate ions tohydroxyl groups in the polyhydroxy compound that is higher than thatfound to be optimal for a given aqueous preservation medium of thepresent invention may, in many circumstances, still result, uponpreservation, in a preserved biological material composition havingimproved structural and functional integrity, such as improved activityon rehydration, storage stability, or advantages in cost or processingover a preserved composition resulting from an aqueous preservationmedium containing only the polyhydroxy compound. Therefore, it ispreferred that the ratio for a given aqueous preservation medium of thepresent invention be less than or equal to that resulting in optimalstability and activity upon rehydration, with the use of the optimumratio being most preferred. However, an aqueous preservation mediumhaving a ratio greater than that needed for optimal activity of thepreserved biological material upon rehydration can be used.

[0053] Other Components

[0054] The protectant mixture and/or aqueous preservation medium of thepresent invention can contain other components. For example, they maycontain a buffer to maintain the pH of the medium at an optimal valuefor a given biological material. It should be noted that the phosphateions in the mixture and/or medium function as a buffer as well, soadditional non-phosphate buffers may not be needed. If a phosphatebuffer is used, the amount of phosphate ions present in the buffershould be included in determining the molar ratio of phosphate ions tohydroxyl groups of the polyhydroxy compound in the mixture and/oraqueous preservation medium, as well as the resulting preservedbiological material composition. The pH at which a given biologicalmaterial is most stable is known in the art. Generally, the preservationmedia of the present invention should have a pH in the range from about5 to about 10, with a pH in the range from about 6 to about 9 being mostpreferred.

[0055] The protectant mixture and/or aqueous preservation medium mayalso contain one or more antioxidants, such as sodium thiosulfate,ascorbic acid, citric acid, and sodium citrate. If an antioxidant isused, it can be present in an amount known to be useful in the art.

[0056] The protectant mixture and/or aqueous preservation medium mayalso contain other components that may act as drying agents and/orosmoprotectants, such as methanol, ethanol, glycerol and DMSO. Thesecomponents tend to reduce residual moisture or balance osmotic stressesin the preserved biological material compositions made from the aqueouspreservation media of the present invention, which may in some casesresult in better storage capability.

[0057] Preparation of the Protectant Mixture, Aqueous PreservationMedium and Preserved Composition of the Present Invention

[0058] The protectant mixture of the present invention can be preparedas follows. The polyhydroxy compound and the source of phosphate ionsare added in the desired proportion to an aqueous solution. It ispreferred that the phosphate ion source be dissolved in the solutionprior to the addition of the polyhydroxy compound. The mixture may beheated if necessary to effect dissolution. The solution should be mixedthoroughly, until a homogenous protectant mixture is obtained. Thisprotectant mixture can then be stored as an aqueous solution, or can besubjected to various processes known in the art to produce a solidmixture, such as a powder. The powder can be anhydrous or a partiallycrystalline hydrated material. Where the protectant mixture is a solid,such as a powder, it should be reconstituted with an aqueous solutionbefore it is used to make the aqueous preservation medium of the presentinvention. However, the solid mixture can be directly added to anaqueous solution containing the biological material to form the aqueouspresent medium.

[0059] The protectant mixture in the form of an aqueous solution is thenadded to an aqueous solution of the biological material. If theprotectant mixture was prepared in an aqueous buffer solution, theaqueous solution of biological materials is preferably prepared in thesame buffer. These two solutions are then thoroughly mixed, to form theaqueous preservation medium of the present invention. If an antioxidantis being used, it is generally added to the aqueous solution ofbiological material before addition of the protectant solution.

[0060] The amount of biological material used in the aqueouspreservation medium of the present invention can be varied as desired,depending for example upon the specific biological material to bepreserved, the amounts of phosphate ions and polyhydroxy compoundpresent, the preservation technique to be employed.

[0061] The aqueous preservation medium can then be preserved by one ormore techniques known in the art, including freezing, freeze-drying,vacuum-drying, ambient, air-drying, and/or spray-drying, to provide apreserved biological material composition of the present invention. Theresulting preserved biological material composition can be anhydrous, orcan be a partially crystalline hydrated material. It is preferred thatthe preserved biological material be substantially anhydrous in nature.Here, “substantially anhydrous” means that the preserved biologicalmaterial composition contains less than 10% water measured by KarlFisher analysis.

EXAMPLE 1

[0062] Various protectant mixtures in aqueous solution form were made inButterfield's buffer (0.6 mM potassium phosphate buffered water having apH of 7.2), by adding predetermined amounts of a polyhydroxy compoundand phosphate ions, and mixing to form a homogenous solution. A givenprotectant mixture was then added to an L. acidophilus cell solution ina 1:1 mass ratio, and the resulting aqueous protective medium wasthoroughly mixed and allowed to incubate at room temperature for 30minutes. The L. acidophilus cell solution was prepared as follows:concentrated L. acidophilus bacterial cultures having a 15.3 wt % drymass were mixed with 1.9% sodium thiosulfate to form an L. acidophiluscell solution and allowed to incubate at room temperature for 30minutes.

[0063] Samples of each aqueous protective medium made in the abovemanner were then subjected to either freezing, freeze-drying orvacuum-drying.

[0064] For the samples that were frozen, the aqueous preservation mediumwas dripped through a 25 gauge syringe needle into liquid nitrogen. Thedrops usually formed pellets of 2-3 mm in diameter, which frozecompletely within about 10 seconds. These samples of preserved cellcomposition were then allowed to thaw in open atmosphere at roomtemperature.

[0065] The samples that were freeze-dried were subjected to freezing asdetailed above, and the resulting pellets were placed in a Virtis 12ELfreeze dryer on precooled shelves at a temperature no greater than −45°C. Additionally, small samples of the preservation medium were weighedto determine their density, and then freeze-dried as a thin layer inglass dishes. These samples were weighed before and after freeze-dryingto calculate the amount of water lost by each sample duringfreeze-drying, and hence to determine the amount of water needed forrehydration.

[0066] All of the freeze-dried samples were subjected to the followingfreeze-drying protocol, where the pressures and temperatures are setpoints. After the samples were placed in the freeze dryer as discussedabove, the freeze dryer was evacuated with a pressure of 0 mtorr. Thesamples were held at −45° C. and 0 mtorr for 1200 minutes, after whichthe temperature and pressure were increased to −40° C. and 20 mtorr,respectively, over a period of 50 minutes. The samples where then heldat −40° C. and 20 mtorr for 600 minutes. The temperature was thenincreased to −35° C. over a period of 50 minutes, and the samples werethen held at −35° C. and 20 mtorr for 600 minutes. The temperature andpressure were next increased to −30° C. and 50 mtorr, respectively, over50 minutes and the samples were then held at −30° C. and 50 mtorr for600 minutes. At the end of that period of time, the temperature andpressure were increased to −25° C. and 100 mtorr, respectively, over 50minutes, and the samples were held at −25° C. and 100 mtorr for 600minutes. The temperature was then increased to −20° C. over 50 minutes,and the samples were then held at −20° C. and 100 mtorr for 600 minutes.The temperature was next increased to −10° C. over 100 minutes, and thesamples were held at that temperature and 100 mtorr for 600 minutes.

[0067] The samples were then increased to a final temperature of 40° C.at a rate of 0.5° C./min, at 50 mtorr. The samples were then held at thefinal temperature at 0 mtorr for 1200-2400 minutes. The freeze dryer wasthen vented with nitrogen gas, and the preserved cell compositionsamples were removed, sealed in 50 ml plastic centrifuge tubes, andstored in a 37° C. incubator.

[0068] For the samples that were subjected to vacuum drying, 0.2 ml ofthe preservation medium was placed in a 1.8 ml plastic microcentrifugetube. The samples were then placed into a Savant Instruments SVC-100HSpeedVac Concentrator vacuum dryer and rotary evaporated at roomtemperature for approximately 4 days at a final pressure of 85 mtorr, toobtain preserved cell compositions. The tubes were then sealed, andstored at room temperature in a desiccator.

[0069] At the time the samples were made and subjected to freezing,vacuum-drying or freeze-drying, additional samples from the aqueouspreservation media were taken, diluted, and plated on agar medium usinga standard pour-plating technique to determine the viable cell number inthe preservation medium prior to any preservation process beingconducted. First, the samples were diluted to achieve a cellconcentration of about 100 cells/ml. One or two milliliters of thediluted preservation medium were then placed in a petri dish and mixedwith liquid agar growth medium (15 g/L agar with 55 g/L MRSLactobacillus Broth and 0.1% L-cysteine) at 45° C. The dish was allowedto cool, solidifying the agar, at which time the dishes were invertedand placed in an anaerobic growth chamber (GasPak jar) at 37° C. for 2-3days, at which time colonies were visible in the agar. In principle,each colony represents a single viable cell in the preservation medium.

[0070] At various times, portions of the preserved cell compositionsprepared by freeze-drying and vacuum drying were rehydrated inButterfield's buffer. Freeze-dried samples were rehydrated toapproximately {fraction (1/100)}^(th) of their original concentration,allowed to incubate for 30 minutes, diluted, and plated as describedabove. Vacuum-dried samples were rehydrated to approximately ⅕^(th)their original concentration, mixed to fully dissolve the pellet,diluted, and plated as described above. All samples were plated in atleast triplicate. For these experiments, time zero is the time at whichthe samples were removed from the dryers. The preserved cellcompositions that were prepared by freezing samples of the preservationmedium in liquid nitrogen were plated as described above after completethawing at room temperature had occurred.

[0071] The results for a given set of samples are set forth below inTable 1, where the numbers represent the percent of original activitythat was recovered upon rehydration. The “solution” column provides theamount of viable cells in a given preservation medium, with the amountof viable cells in the “cells alone” sample being defined as 100%. TABLE1 Vacuum Dried Freeze Dried Sample Solution Frozen Time = 0 9 days 27days Time = 0 9 days 28 days 64 days 101 days Cells Alone 100 105 0 0 NA³ 56 0 NA NA NA 20% Trehalose + 96 97 10 7 NA 44 31 21  5 NA thio 20%Trehalose + 83 93 27 26 17 69 16 14  6 NA borate (0.3)¹ + thio² 20%Trehalose + 89 88 12 6 NA 55 28 13  3 NA NaH₂PO₄ (0.1) + thio 20%Trehalose + 94 88 5 1 NA 45 24  7  0 NA NaH₂PO₄ (0.3) + thio 20%Trehalose + 77 95 3 2 NA 51 26  0 NA NA NaH₂PO₄ (0.5) + thio 20%Trehalose + 92 86 3 2 NA 71 4 NA NA NA NaH₂PO₄ (1.0) + thio 20%Trehalose + 103 105 51 48 39 84 72 58 17  0 thio + NaH₂PO₄/ K₂HPO₄ (1.0)pH 5.6 20% Trehalose + 106 107 4 3 NA 82 65 24  0 NA thio + NaH₂PO₄/K₂HPO₄ (1.0), pH 4.9 20% Trehalose + 93 106 52 59 73 82 76 70 67 57thio + NaH₂PO₄/ K₂HPO₄ (1.0), pH 6.6 20% Trehalose + 96 104 23 18  5 8770 47  7 NA thio + NaH₂PO₄/ Na₂HPO₄ (1.0) pH 5.3

EXAMPLE 2

[0072] The procedure of Example 1 was repeated, using phosphate,carbonate, or sulfate ions with polyhydroxy compounds in varyingamounts. The results for a given set of samples are set forth below inTable 2, where the numbers represent the percent of original activitythat was recovered upon rehydration. The “solution” column provides theamount of viable cells in a given preservation medium, with the amountof viable cells in the “cells alone” sample being defined as 100%. TABLE2 Vacuum Dried Freeze Dried⁴ Sample Solution Frozen Time = 0 19 days 34days Time = 0 19 days 38 days 74 days 99 days Cells Alone 100 102 2 46 0NA NA NA 20% Trehalose + 83 88 14 18  NA⁴ 52 13 13  5 NA thio 20%Sucrose + 100 103 4 NA NA 80 33 19 20 15 thio 20% Trehalose + 101 90 3721 21 49 0 NA NA NA borate(0.3)¹ + thio² 20% Sucrose + 93 108 35 27 NA55 3 NA NA NA borate(0.3) + thio 20% 1 59 0 NA NA 27 8 NA NA NATrehalose³ + Na₂CO₃ (1.0) + thio 20% Sucrose³ + 1 34 0 NA NA 16 0 NA NANA Na₂CO₃ (1.0) + thio 20% Trehalose + 87 92 24 21 18 73 1 NA NA NANa₂SO₄ (0.75) + thio 20% Sucrose + 91 86 8 NA NA 66 7 NA NA NA Na₂SO₄(0.75) + thio 20% Trehalose + 85 86 65 61 68 74 54 46 37 26 thio +NaH₂PO₄/ K₂HPO₄(1.0), pH 6.5 20% Sucrose + 93 90 51 34 NA 74 43 29 27 12thio + NaH₂PO₄/ K₂HPO₄(1.0), pH 6.5

EXAMPLE 3

[0073] The procedure of Example 1 was repeated, using preservation mediacontaining various sources of phosphate ions in varying amounts, andvarying amounts of trehalose, with the exception that the freeze-driedsamples were dried to 50° C. In addition, the residual water in thefreeze-dried samples was measured by Karl Fischer (KF) analysis, and theglass transition temperature (T_(g)) was obtained. The results for agiven set of samples are set forth below in Table 3, where the numbersrepresent the percent of original activity that was recovered uponrehydration. The “solution” column provides the amount of viable cellsin a given preservation medium, with the amount of viable cells in the“cells alone” sample being defined as 100%. TABLE 3 Vacuum Dried FreezeDried % water T_(g) 19 95 21 48 92 Sample by KF (° C.) Solution FrozenTime = 3 days days Time = 0 days days days Cells Alone 0.8 47 100 10 0NA NA 22 0 NA NA 20% Trehalose + 0.7 88 105 104 9 NA NA 50 10 NA NA thio20% Trehalose + 5.3 32 * 105 68 78 50 44 27 15  5 thio² + NaH₂PO₄/K₂HPO₄(0.5)¹, ph 6.6 20% Trehalose + 3.2 85 105 114 75 89 70 66 63 55 48thio + NaH₂PO₄/ K₂HPO₄(1), pH 6.6 20% Trehalose + 1.6 46  87 87 51 59  569 41 27 13 thio + NaH₂PO₄/ K₂HPO₄(1), pH 6.6 + Methanol 20% Trehalose +3.5 82  97 110 70 63 54 70 60 61 49 thio + KH₂PO₄/ K₂HPO₄(1), pH 6.6 5%Trehalose + 3.8 44 108 110 35 19 27 51 26  9 NA thio + NaH₂PO₄/K₂HPO₄(1), pH 6.6

EXAMPLE 4

[0074] The procedure of Example 1 was repeated, using varying amounts oftrehalose and different phosphate ion sources in the preservationmedium, with the exception that the freeze-dried samples were dried to atemperature of 50° C. In two samples, ethanol was added as a dryingagent to the preservation medium, while in one sample, the L.acidophilus cells were “washed” (centrifuged, decanted and resuspended)to remove residual growth medium. Media containing different bufferswere also tested. The results for a given set of samples are set forthbelow in Table 4, where the numbers represent the percent of originalactivity that was recovered upon rehydration. The “solution” columnprovides the amount of viable cells in a given preservation medium, withthe amount of viable cells in the “cells alone” sample being defined as100%. TABLE 4 Vacuum Dried Freeze Dried³ Sample Solution Frozen 40 days68 days Time = 0 14 days 40 days 68 days Cells Alone 100 116 0 NA 36 0NA 20% Trehalose + thio² 89 98 22  8 73 28  4 17.5% Trehalose + thio +101 120 41 55 65 85 23 22 7 KH₂PO₄/K₂HPO₄(1)¹, pH 6.5 17.5% Trehalose +thio + 98 112 0 NA 48 9 NA KH₂PO₄/K₂HPO₄(1), pH 6.5 + Ethanol (1 mol/molP) 17.5% Trehalose + thio + 89 95 0 NA 14 0 NA KH₂PO₄/K₂HPO₄(1), pH6.5 + Ethanol (2 mol/mol P) 20% Trehalose + thio + 106 97 55 44 30 77 5063 54 K₂HPO₄(1) 20% Trehalose + thio + 59 49 4 NA 47 15 27 29K₂HPO₄(1) + citric acid/NH3OH buffering 20% Trehalose + thio + 59 48 3NA 27 6 NA K₂HPO₄(1) + lactic acid/NH3OH buffering 17.5% Trehalose +thio + 97 122 40 41 24 80 30 20 6 KH₂PO₄/K₂HPO₄(1), pH 6.5 Washed Cells

EXAMPLE 5

[0075] The procedure of Example 1 was repeated, using trehalose,phosphate ions, and various antioxidants or no antioxidant, with theexception that the samples were freeze-dried to a temperature of 50° C.The results for a given set of samples are set forth below in Table 5,where the numbers represent percent of original activity that wasrecovered upon rehydration. The “solution” column provides the amount ofviable cells in a given preservation medium, with the amount of viablecells in the “cells alone” sample being defined as 100%. TABLE 5 VacuumDried Freeze Dried Sample Solution Frozen Time = 0 9 days 25 days Time =0 9 days 25 days Cells Alone 100 100 0 0 0 1 0 0 20% Trehalose + 99 8525 30 15 65 51 33 KH₂PO₄/K₂HPO₄(1)^(1, 3) 20% Trehalose + 80 71 46 35 2073 42 19 KH₂PO₄/K₂HPO₄(1)^(1, 3) + thio² 20% Trehalose + 95 82 33 30 1661 38 25 KH₂PO₄/K₂HPO₄(1)^(1, 3) + ascorbic acid 20% Trehalose + 80 7516 24 11 49 17 3 KH₂PO₄/K₂HPO₄(1)^(1, 3) + citric acid 20% Trehalose +88 76 42 24 10 74 46 12 KH₂PO₄/K₂HPO₄(1)^(1, 3) + sodium citrate

EXAMPLE 6

[0076] The procedure of Example 1 was repeated, with the exceptions thata Pediococcus species was substituted for L. acidophilus cells, and thefreeze-dried samples were dried to a temperature of 25° C. or furtherdried to 50° C. The results for a given set of samples are set forthbelow in Table 6, where the numbers represent percent of originalactivity that was recovered upon rehydration. The “solution” columnprovides the amount of viable cells in a given preservation medium, withthe amount of viable cells in the “cells alone+thio” sample beingdefined as 100%. TABLE 6 Freeze Dried Freeze Dried Vacuum DriedT_(final) = 25 C. T_(final) = 50 C. T_(final) = 25 C. T_(final) = 50 C.Sample Solution Frozen Time = 0 31 days 60 days Time = 0 Time = 0 31days 60 days 31 days 59 days Cells 100 118 73 62 75 96 73 5 2 Alone +thio 20% 121 121 84 61 32 117 115 55 19 48 25 Trehalose + thio² 20% 114113 104 95 89 100 113 96 78 97 97 Trehalose + thio +KH₂PO₄/K₂HPO₄(1)^(1, 3) 20% 102 122 99 93 81 118 94 75 65 88 81Trehalose + KH₂PO₄/K₂PO₄(1)^(1, 3) + 2% Citric Acid

EXAMPLE 7

[0077]_(L)-Lactate dehydrogenase (LDH, EC 1.1.1.27, Type II, rabbitmuscle) was dialyzed overnight at 4° C. in 100 mM potassium phosphatebuffer solution at pH 7.5. The total protein content was assayed usingSIGMA DIAGNOSTIC, a protein determination kit purchased from SigmaChemical Company (St. Louis, Mo.), using the modified biuret method ofOhnishi and Barr, “Simplified Method for Quantitating Protein using theBiuret and Phenol Reagents,” Analytical Biochem. 86:193-200 (1978). Theprotein assay was conducted at the characteristic absorption at 725 nmat room temperature using a Varian UV Spectrophotometer. The reactionmixture contained 100 mM potassium phosphate buffer (pH 7.5), 0.150 mMNADH, and 1.20 mM pyruvic acid.

[0078] To prepare the samples, the dialyzed LDH was diluted with thesame potassium phosphate buffer that had been used for dialysis. Theresulting enzyme solution had a phosphate ion concentration of 100 mM,and a LDH concentration of 50 μg/ml. Three sets of protectant mixtureswere then made. The mixture sets had a trehalose concentration of 200mM, 400 mM and 600 mM, respectively. Each mixture set consisted of fourseparate protectant samples, containing phosphate ions in aconcentration of 100 mM, 300 mM, 500 mM and 700 mM, respectively. Thesesamples were made by dissolving the trehalose in an aqueous phosphatesolution containing a given amount of phosphate ions.

[0079] Two milliliters of the LDH solution were then mixed with 2 ml ofeach of the twelve protectant mixtures to provide 4 ml solutions ofaqueous preservation media having a trehalose concentration of either100 mM, 200 mM, or 300 MM, LDH concentration of 25 μg/ml, and phosphateion concentrations of 100 mM, 200 mM, 300 mM or 400 mM. The above samplepreparation is shown in Table 7. TABLE 7 Sample 1 2 3 4 (Set 1) Enzyme50 μg/ml LDH 50 μg/ml LDH 50 μg/ml LDH 50 μg/ml LDH Solution 100 mMPhosphate 100 mM Phosphate 100 mM Phosphate 100 mM PhosphateCryoprotective 200 mM Trehalose 200 mM Trehalose 200 mM Trehalose 200 mMTrehalose Solution 100 mM Phosphate 100 + 200 mM Phosphate 100 + 400 mMPhosphate 100 + 600 mM Phosphate Mixture 25 μg/ml LDH 25 μg/ml LDH 25μg/ml LDH 25 μg/ml LDH Solution 100 mM Trehalose 100 mM Trehalose 100 mMTrehalose 100 mM Trehalose 100 mM Phosphate 200 mM Phosphate 300 mMPhosphate 400 mM Phosphate (Set 2) Enzyme 50 μg/ml LDH 50 μg/ml LDH 50μg/ml LDH 50 μg/ml LDH Solution 100 mM Phosphate 100 mM Phosphate 100 mMPhosphate 100 mM Phosphate Cryoprotective 400 mM Trehalose 400 mMTrehalose 400 mM Trehalose 400 mM Trehalose Solution 100 mM Phosphate100 + 200 mM Phosphate 100 + 400 mM Phosphate 100 + 600 mM PhosphateMixture 25 μg/ml LDH 25 μg/ml LDH 25 μg/ml LDH 25 μg/ml LDH Solution 200mM Trehalose 200 mM Trehalose 200 mM Trehalose 200 mM Trehalose 100 mMPhosphate 200 mM Phosphate 300 mM Phosphate 400 mM Phosphate (Set 3)Enzyme 50 μg/ml LDH 50 μg/ml LDH 50 μg/ml LDH 50 μg/ml LDH Solution 100mM Phosphate 100 mM Phosphate 100 mM Phosphate 100 mM PhosphateCryoprotective 600 mM Trehalose 600 mM Trehalose 600 mM Trehalose 600 mMTrehalose Solution 100 mM Phosphate 100 + 200 mM Phosphate 100 + 400 mMPhosphate 100 + 600 mM Phosphate Mixture 25 μg/ml LDH 25 μg/ml LDH 25μg/ml LDH 25 μg/ml LDH Solution 300 mM Trehalose 300 mM Trehalose 300 mMTrehalose 300 mM Trehalose 100 mM Phosphate 200 mM Phosphate 300 mMPhosphate 400 mM Phosphate

[0080] Forty-eight vials were then prepared for each of the abovesamples of preservation media. Each vial was labeled and weighed, and 1ml of preservation medium was pipetted into each vial. Each vial wasthen reweighed. The samples were then frozen by immersion of the vialsin liquid nitrogen (“quench”) or by placement in the freeze-dryer onprecooled shelves at a temperature no greater than −45° C. (“slowfreeze”). All of the freeze-dried samples were subjected to thefollowing freeze-drying protocol in a Virtis 12EL freeze-dryer, whereall pressures and temperatures are set points. After the samples wereplaced in the freeze-dryer as discussed above, the freeze-dryer wasevacuated with a pressure of 0 mtorr. The samples were held at −45° C.and 0 mtorr for 600 minutes, after which the temperature and pressurewere increased to −40° C. and 20 mtorr, respectively, over a period of50 minutes. The samples where then held at −40° C. and 20 mtorr for 600minutes. The temperature was then increased to −35° C. over a period of50 minutes, and the samples were then held at −35° C. and 20 mtorr for600 minutes. The temperature and pressure were next increased to −30° C.and 50 mtorr, respectively, over 50 minutes and the samples were thenheld at −30° C. and 50 mtorr for 600 minutes. At the end of that periodof time, the temperature and pressure were increased to −25° C. and 100mtorr, respectively, over 50 minutes, and the samples were held at −25°C. and 100 mtorr for 600 minutes. The temperature was then increased to−20° C. over 50 minutes, and the samples were then held at −20° C. and100 mtorr for 600 minutes. The temperature was next increased to −10° C.over 100 minutes, and the samples were held at that temperature and 100mtorr for 600 minutes.

[0081] The samples were then increased to a temperature of 25° C. at 50mtorr over a period of 700 minutes. The samples were then held at thefinal temperature at 0 mtorr for 2140 minutes until unloading. Thefreeze dryer was then vented with nitrogen gas, and the preserved LDHsamples in the vials were removed and measured for LDH activity. Beforethe activity was measured, each sample was weighed to determine theamount of water loss, and rehydrated with purified water (MilliporeSystem from Millipore Corp.) in the amount of the water that was lost.LDH activity was then determined using the same method discussedpreviously.

[0082] The results for a given set of samples are set forth in FIG. 1(freeze-drying) and FIG. 2 (freezing).

EXAMPLE 8

[0083]_(L)-Lactate dehydrogenase (LDH, EC 1.1.1.27, Type II, rabbitmuscle) was dialyzed overnight at 4° C. in 100 mM potassium phosphatebuffer solution at pH 7.5. The total protein content was assayed usingSIGMA DIAGNOSTIC, a protein determination kit purchased from SigmaChemical Company (St. Louis, Mo.), using the modified biuret method ofOhnishi and Barr, “Simplified Method for Quantitating Protein using theBiuret and Phenol Reagents,” Analytical Biochem. 86:193-200 (1978). Theprotein assay was conducted at the characteristic absorption at 725 nmat room temperature using a Varian UV Spectrophotometer. The reactionmixture contained 100 mM potassium phosphate buffer (pH 7.5), 0.150 mMNADH, and 1.20 mM pyruvic acid.

[0084] To prepare the samples, LDH was added to four 50 ml containersand diluted with the same potassium phosphate buffer that had been usedfor dialysis to make a 25 ml solution. In each of the samples, theenzyme concentration was 50 μg/ml. Four protectant mixtures having avolume of 25 ml were then prepared in 50 ml containers. Each mixturecontained 400 mM of trehalose, and varying amounts of phosphate ion. Tomake the first mixture (reference), the trehalose was dissolved in 10 mMpotassium phosphate solution. For the second mixture, the trehalose wasdissolved in 100 mM potassium phosphate solution. The third and fourthmixtures were made by dissolving the trehalose in 500 mM potassiumphosphate solution and 900 mM potassium phosphate solution,respectively.

[0085] The LDH samples were then mixed with the protectant mixtures toprovide 50 ml solutions of aqueous preservation media having a LDHconcentration of 25 μg/ml, a trehalose concentration of 200 mM andvarying LDH and phosphate ion concentrations.

[0086] The phosphate ion concentration for samples 1-4 was 10 mM, 100mM, 300 mM, and 500 mM, respectively, for a phosphate ion to trehalosemolar ratio of 0.05 for Sample 1, 0.5 for Sample 2, 1.5 for Sample 3,and 2.5 for Sample 4. The above sample preparation is shown below inTable 8. TABLE 8 Sample 1 2 3 4 LDH 50 μg/ml LDH 50 μg/ml LDH 50 μg/mlLDH 50 μg/ml LDH Solution 10 mM phosphate 100 mM phosphate 100 mMphosphate 100 mM phosphate Protectant 400 mM trehalose 400 mM trehalose400 mM trehalose 400 mM trehalose Mixture 10 mM phosphate 100 mMphosphate 500 mM phosphate 900 mM phosphate Preservation 25 μg/ml LDH 25μg/ml LDH 25 μg/ml LDH 25 μg/ml LDH Medium 200 mM trehalose 200 mMtrehalose 200 mM trehalose 200 mM trehalose 10 mM phosphate 100 mMphosphate 300 mM phosphate 500 mM phosphate

[0087] Forty vials were then prepared for each of the above four samplesof preservation media. Each vial was labeled and weighed, and 1 ml ofpreservation medium was pipetted into each vial. Each vial was thenreweighed. The samples were then freeze-dried using the same protocol asdescribed in Example 7.

[0088] After freeze-drying was complete, the preserved LDH samples inthe vials were removed, the vials were sealed, and stored in either a37° C. incubator, a 30° C. incubator or at 4° C. in a refrigerator.

[0089] LDH activity was then measured periodically for the samples.Before the activity was measured, each sample was weighed to determinethe amount of water loss, and rehydrated with purified water (MilliQSystem from Millipore Corp.) in the amount of the water that was lost.LDH activity was then determined using the same method discussedpreviously.

[0090] The results for a given set of samples are set forth in FIGS.3-5, where “Z” is the molar ratio of phosphate ions to trehalose.

EXAMPLE 9

[0091] The procedure of Example 1 was repeated. The results are setforth below in Table 9, where the numbers represent percent of originalactivity that was recovered upon rehydration. The “solution” columnprovides the amount of viable cells in a given preservation medium, withthe amount of viable cells in the “cells alone” sample being defined as100%. TABLE 9 Freeze Dried Sample Solution Frozen T = 3 days Cells Alone100  105   0 20% Trehalose 91 89 11 20% Trehalose + thio² 89 77  8 20%Trehalose + KH₂PO₄/ 72 78 39 K₂HPO₄(1)¹, pH 6.4 20% Trehalose + thio +KH₂PO₄/ 54 61 41 K₂HPO₄(1), pH 6.4 Cells + KHPO₄/K₂HPO₄ (Same 89 90  7phosphate per g cells as Samples 4, 5 above) 20% Trehalose + thio +K₂HPO₄(1) 75 81 39

EXAMPLE 10

[0092] Aqueous protectant mixtures containing 7.5% of sucrose ortrehalose on a molar basis and varying amounts of phosphate ionsprovided by either potassium monophosphate or potassium diphosphate wereprepared, and 25 microliters of each mixture was sealed into an aluminumDifferential Scanning Calorimetry pan. The samples were then quenched byimmersion in liquid nitrogen and loaded into the Differential ScanningCalorimeter, which had been precooled to −140° C. The samples were thenscanned at a rate of 5° C./min to a temperature of 50° C., and the glasstransition temperature (T_(g)) was determined. The results for eachsample are shown in FIG. 6.

We claim:
 1. An aqueous preservation medium for preserving biologicalmaterials, comprising: (a) a biological material; (b) at least onepolyhydroxy compound, where the total amount of polyhydroxy compound inthe medium is from about 5% to about 60% by weight of the medium; and(c) phosphate ions, where the total amount of phosphate ions in themedium is such that the molar ratio of phosphate ions to hydroxyl groupsin the polyhydroxy compound is from about 0.025 to about 0.625.
 2. Theaqueous preservation medium of claim 1, where the pH of the medium isfrom about 5 to about
 10. 3. The aqueous preservation medium of claim 1,where the polyhydroxy compound is selected from a group consisting ofmonosaccharides, disaccharides, and polysaccharides.
 4. The aqueouspreservation medium of claim 3, where the polyhydroxy compound istrehalose.
 5. The aqueous preservation medium of claim 1, where thetotal amount of polyhydroxy compound in the medium is from about 10% toabout 30% by weight of the medium.
 6. The aqueous preservation medium ofclaim 3, where the total amount of polyhydroxy compound in the medium isfrom about 10% to about 30% by weight of the medium.
 7. The aqueouspreservation medium of claim 1, where the molar ratio of phosphate ionsto hydroxyl groups in the polyhydroxy compound is from about 0.0375 toabout 0.625.
 8. The aqueous preservation medium of claim 1, where thebiological material is selected from the group consisting of cells,proteins, and enzymes.
 9. An aqueous preservation medium for preservingbiological materials, comprising: (a) a biological material; (b)trehalose, where the trehalose is present in an amount from about 5% toabout 60% by weight of the medium; and (c) phosphate ions, where thetotal amount of phosphate ions in the medium is such that the molarratio of phosphate ions to trehalose is from about 0.2 to about
 5. 10.The aqueous preservation medium of claim 9, where the trehalose ispresent in an amount from about 10% to about 30% by weight of themedium.
 11. The aqueous preservation medium of claim 10, where the pH isfrom about 5 to about
 10. 12. The aqueous preservation medium of claim9, where the molar ratio of phosphate ions to trehalose is from about0.3 to about
 5. 13. The aqueous preservation medium of claim 9, wherethe biological material is selected from the group consisting of cells,proteins, and enzymes.
 14. A method of preparing a preserved biologicalmaterial composition, comprising: (a) forming an aqueous preservationmedium comprising (i) a biological material; (ii) at least onepolyhydroxy compound, where the total amount of polyhydroxy compound inthe medium is from about 5% to about 60% by weight of the medium; and(iii) phosphate ions, where the total amount of phosphate ions in themedium is such that the molar ratio of phosphate ions to moles ofhydroxyl groups in the polyhydroxy compound is from about 0.025 to about0.625; and (b) preserving the aqueous preservation medium using at leastone preservation process.
 15. The method of claim 14, where thepreservation processes are one or more processes selected from the groupconsisting of freezing, freeze-drying, ambient-air drying,vacuum-drying, and spray drying.
 16. The method of claim 14, where thepH of the medium is from about 5 to about
 10. 17. The method of claim14, where the polyhydroxy compound is selected from a group consistingof monosaccharides, disaccharides, and polysaccharides.
 18. The methodof claim 14, where the polyhydroxy compound is trehalose.
 19. The methodof claim 14, where the total amount of polyhydroxy compound in themedium is from about 10% to about 30% by weight of the medium.
 20. Themethod of claim 17, where the total amount of polyhydroxy compound inthe medium is from about 10% to about 30% by weight of the medium. 21.The method of claim 14, where the molar ratio of phosphate ions to molesof hydroxyl groups in the polyhydroxy compound is from about 0.0375 toabout 0.625.
 22. The method of claim 14, where the biological materialis selected from the group consisting of cells, proteins, and enzymes.23. A method of preparing a preserved biological material compositioncomprising: (a) forming an aqueous preservation medium comprising (i) abiological material; (ii) trehalose, where the total amount ofpolyhydroxy compound in the medium is from about 5% to about 60% byweight of the medium; and (iii) phosphate ions, where the total amountof phosphate ions in the medium is such that the molar ratio ofphosphate ions to trehalose is from about 0.2 to about 5; and (b)preserving the aqueous preservation medium using at least onepreservation process.
 24. The method of claim 23, where the preservationprocesses are one or more processes selected from the group consistingof freezing, freeze-drying, ambient-air drying, vacuum-drying, and spraydrying.
 25. The method of claim 23, where the trehalose is present in anamount from about 10% to about 30% by weight of the medium.
 26. Themethod of claim 25, where the pH is from about 5 to about
 10. 27. Themethod of claim 26, where the molar ratio of phosphate ions to trehaloseis from about 0.3 to about
 5. 28. The method of claim 23, where thebiological material is selected from the group consisting of cells,proteins, and enzymes.
 29. A protectant mixture in solid form for use inpreserving biological materials, comprising: (a) at least onepolyhydroxy compound, where the total amount of polyhydroxy compound inthe mixture is from about 10% to about 95% by weight of the mixture; and(b) phosphate ions, where the total amount of phosphate ions in themixture is such that the molar ratio of phosphate ions to hydroxylgroups in the polyhydroxy compound is from about 0.025 to about 0.625.30. The protectant mixture of claim 29, where the total amount ofpolyhydroxy compound in the mixture is from about 20% to about 95% byweight of the mixture.
 31. The protectant mixture of claim 29, where thepolyhydroxy compound is selected from a group consisting ofmonosaccharides and polysaccharides.
 32. The protectant mixture of claim31, where the polyhydroxy compound is trehalose.
 33. The protectantmixture of claim 29, where the molar ratio of phosphate ions to hydroxylgroups in the polyhydroxy compound is from about 0.0375 to about 0.625.34. A protectant mixture in solid form for use in preserving biologicalmaterials, comprising: (a) trehalose, where the trehalose is present inthe mixture in an amount from about 10% to about 95% by weight of themixture; and (b) phosphate ions, where the total amount of phosphateions in the mixture is such that the molar ratio of phosphate ions totrehalose is from about 0.2 to about
 5. 35. The protectant mixture ofclaim 34, where the trehalose is present in an amount from about 20% toabout 95% by weight of the mixture.
 36. The protectant mixture of claim34, where the molar ratio of phosphate ions to trehalose is from about0.3 to about
 5. 37. A protectant mixture in the form of an aqueoussolution, comprising: (a) at least one polyhydroxy compound, where thetotal amount of polyhydroxy compound in the mixture is from about 5% toabout 60% by weight of the mixture; and (b) phosphate ions, where thetotal amount of phosphate ions in the mixture is such that the molarratio of phosphate ions to hydroxyl groups in the polyhydroxy compoundis from about 0.025 to about 0.625.
 38. The protectant mixture of claim36, where the total amount of polyhydroxy compound in the mixture isfrom about 10% to about 30% by weight of the mixture.
 39. The protectantmixture of claim 37, where the polyhydroxy compound is selected from agroup consisting of monosaccharides and polysaccharides.
 40. Theprotectant mixture of claim 39, where the polyhydroxy compound istrehalose.
 41. The protectant mixture of claim 37, where the molar ratioof phosphate ions to hydroxyl groups in the polyhydroxy compound is fromabout 0.0375 to about 0.625.
 42. A protectant mixture in the form of anaqueous solution, comprising: (a) trehalose, where the trehalose ispresent in the mixture in an amount from about 5% to about 60% by weightof the mixture; and (b) phosphate ions, where the total amount ofphosphate ions in the mixture is such that the molar ratio of phosphateions to trehalose is from about 0.2 to about
 5. 43. The protectantmixture of claim 42, where the trehalose is present in an amount fromabout 10% to about 30% by weight of the mixture.
 44. The protectantmixture of claim 42, where the molar ratio of phosphate ions totrehalose is from about 0.3 to about
 5. 45. A preserved biologicalmaterial composition made by the method of claim 14 or claim 23.